Structural and functional characterization of Entamoeba histolytica malic enzyme

Entamoeba histolytica is an amitochondriate protozoan parasite causing amoebiasis in tropical and subtropical countries where sanitary condition is poor. Metronidazole (Mtz) the best recommended and potent drug against amoebiasis needs activation inside the cell. Different enzymes like Pyruvate ferridoxin oxidoreductase (PFOR), nitroreductase and thioredoxin reductase have been found to be involved in the activation of the drug in E. histolytica. Malic enzyme (ME), which has been reported to activate Mtz in T. vaginalis is also present in Entamoeba. To investigate the role of malic enzyme in E. histolytica, the EhME gene has been PCR amplified using genomic DNA of Entamoeba, cloned and expressed in bacterial system as His-tag fusion protein. Enzyme activity of recombinant EhME (rEhME) was measured using NADP and L-malate as substrate. The Km and Vmax for L-malate was found to be 4.2mM and 0.41 mmolmin-1 and Km and Vmax for NADP was found to be 69.6μM and 0.38mmolmin-1 respectively. Ovreexpressed rEhME in E.coli (strain JM109) has been able to increase the susceptibility 5-6 times more than that of control cell (JM109-empty vector) against Mtz. RT-PCR and western blot using anti EhME rabbit polyclonal antibody reveal that upon exposure to Mtz, the expression of ME increases in E. histolytica. ME which is secreted to the extracellular environment was also detected by western blot. The released protein has the capacity to bind in both colonic cells and Entamoeba itself. Binding of native and recombinant EhME (labeled with FITC) to membrane of colonic cells were confirmed by confocal microscope.
To analyse the structural aspect of EhME, two step purification of expressed protein was performed and crystallization was done by sitting drop method using 1.5 M (NH4)2SO4, 0.1M CH3COONa pH 5.0, 12% (v/v) glycerol. A complete diffraction data was collected to 2.2Å resolution using the Rigaku Micromax007 CuKα X-ray generator and Rigaku Raxis IV++ detector. The structure has been solved by single isomorphous replacement with anomalous scattering (SIRAS), as molecular replacement theory failed to determine the structure.
Structural analysis shows that, EhME is a dimeric protein with five domains in each monomer. Rossamman’s folds are present within the domain C and domain D which bears the active site and cofactor binding sites. The structure also favours strong specificity for NADP. This is the first reported structure of malic enzyme among the protozoan parasite.

Keywords: Entamoeba histolytica, Malic enzyme, Drug activation, Pathogenesis, Crystal structure.